REPORT Recurrent de novo missense variants across multiple histone H4 genes underlie a neurodevelopmental syndrome Authors Federico Tessadori, Karen Duran, Karen Knapp, ..., Peter D. Mace, Louise S. Bicknell, Gijs van Haaften Correspondence louise.bicknell@otago.ac.nz (L.S.B.), g.vanhaaften@umcutrecht.nl (G.v.H.) Tessadori et al., 2022, The American Journal of Human Genetics 109, 750– 758 April 7, 2022 Ó 2022 The Author(s). https://doi.org/10.1016/j.ajhg.2022.02.003 ll (Affiliations continued on next page) REPORT Recurrent de novo missense variants across multiple histone H4 genes underlie a neurodevelopmental syndrome Federico Tessadori,1 Karen Duran,2 Karen Knapp,3 Matthias Fellner,3 Deciphering Developmental Disorders Study, Sarah Smithson,4 Ana Beleza Meireles,4 Mariet W. Elting,5 Quinten Waisfisz,5 Anne O’Donnell-Luria,6,7,8 Catherine Nowak,8 Jessica Douglas,8 Anne Ronan,9 Theresa Brunet,10,11 Urania Kotzaeridou,12 Shayna Svihovec,13 Margarita S. Saenz,13 Isabelle Thiffault,14,15,16 Florencia Del Viso,15,16 Patrick Devine,17 Shannon Rego,17 Jessica Tenney,18 Arie van Haeringen,19 Claudia A.L. Ruivenkamp,19 Saskia Koene,19 Stephen P. Robertson,20 Charulata Deshpande,21 Rolph Pfundt,22 Nienke Verbeek,23 Jiddeke M. van de Kamp,7 Janneke M.M. Weiss,7,22 Anna Ruiz,24 Elisabeth Gabau,25 Ehud Banne,26 Alexander Pepler,27 Armand Bottani,28 Sacha Laurent,29 Michel Guipponi,29 Emilia Bijlsma,19 Ange-Line Bruel,30,31 Arthur Sorlin,32 Mary Willis,33 Zoe Powis,34 Thomas Smol,35 Catherine Vincent-Delorme,36 Diana Baralle,37 Estelle Colin,38 Nicole Revencu,39 (Author list continued on next page) Summary Chromatin is essentially an array of nucleosomes, each of which consists of the DNA double-stranded fiber wrapped around a histone octamer. This organization supports cellular processes such as DNA replication, DNA transcription, and DNA repair in all eukaryotes. Human histone H4 is encoded by fourteen canonical histone H4 genes, all differing at the nucleotide level but encoding an invariant protein. Here, we present a cohort of 29 subjects with de novo missense variants in six H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11) identified by whole-exome sequencing and matchmaking. All individuals present with neurodevelopmental features of intellec- tual disability and motor and/or gross developmental delay, while non-neurological features are more variable. Ten amino acids are affected, six recurrently, and are all located within the H4 core or C-terminal tail. These variants cluster to specific regions of the core H4 globular domain, where protein-protein interactions occur with either other histone subunits or histone chaperones. Functional consequences of the identified variants were evaluated in zebrafish embryos, which displayed abnormal general development, defective head organs, and reduced body axis length, providing compelling evidence for the causality of the reported disorder(s). While multiple developmental syndromes have been linked to chromatin-associated factors, missense-bearing histone variants (e.g., H3 oncohistones) are only recently emerging as a major cause of pathogenicity. Our findings establish a broader involvement of H4 variants in develop- mental syndromes. Histones are among the most slowly evolving genes in eu- matic variation in H4 is potentially relevant in a cancer karyotes.1,2 Histone H4 acts as a functional unit by forming setting,7,8 although described variation impacts multiple a dimer with H3, but unlike H3, there are no variant isoforms residues in contrast to the highly recurrent pathogenic linked to specific cellular processes. We previously reported variants observed for the established oncohistone, H3.8 four individuals with a primordial dwarfism phenotype This could be because while the functional importance of with de novo missense variants affecting Lys91 in H4C3 PTMs on N-tails of H3 is linked directly to the chromatin (HIST1H4C; MIM: 602827) and H4C11 (HIST1H4J; MIM: effectors targeting these sites,9–11 variants affecting the his- 602826), a critical residue near the C terminus of the protein tone core (H4 in this case) have more direct and global con- prone to post-translational modifications (PTMs) such as sequences on chromatin organization, as they impact nucle- acetylation3 or, more important here, ubiquitination.4–6 So- osome structure and dynamics.12 1 Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 Utrecht, the Netherlands; 2Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, 3584 Utrecht, the Netherlands; 3Department of Biochemistry, University of Otago, Dunedin 9016, New Zealand; 4Bristol Regional Genetics Service, University Hospitals Bristol and Weston NHS Foundation Trust, Bristol BS2 8EG, UK; 5Amsterdam UMC, Afdeling Klinische genetica, 1081 Amsterdam, the Netherlands; 6Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; 7Division of Genetics and Genomics, Boston Children’s Hospital, Boston, MA 02115, USA; 8Manton Cen- ter for Orphan Disease Research, Boston Children’s Hospital, Boston, MA 02115, USA; 9Clinical Genetics, Hunter Genetics Unit, Waratah, NSW 2298, Australia; 10Institute of Medical Genetics, 81675 Munchen, Germany; 11Institute of Neurogenomics, Helmholtz Zentrum München, 85764 Neuherberg, Germany; 12Division of Child Neurology and Inherited Metabolic Diseases, Department of Pediatrics, Heidelberg University Hospital, 69120 Heidelberg, Germany; 13Section of Genetics and Metabolism, Department of Pediatrics, The Children’s Hospital Colorado, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; 14University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108, USA; 15Center for Genomic Medicine, Children’s Mercy Research Institute, Kansas City, MO 64108, USA; 16Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas (Affiliations continued on next page) Ó 2022 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 750 The American Journal of Human Genetics 109, 750–758, April 7, 2022 Eduardo Calpena,40 Andrew O.M. Wilkie,40 Maya Chopra,41 Valerie Cormier-Daire,42 Boris Keren,43 Alexandra Afenjar,44 Marcello Niceta,45 Alessandra Terracciano,46 Nicola Specchio,47 Marco Tartaglia,45 Marlene Rio,48 Giulia Barcia,48 Sophie Rondeau,48 Cindy Colson,49 Jeroen Bakkers,1,50 Peter D. Mace,3 Louise S. Bicknell,3,* and Gijs van Haaften2,* We have identified an additional cohort of 29 individ- in different H4 genes. The absence of any substitutions at uals with de novo missense variants in six different histone Lys91 could reflect a stronger requirement for fidelity at H4 genes: H4C3 (HIST1H4C), H4C4 (HIST1H4D; MIM: this position, especially given the post-translation modifi- 602823), H4C5 (HIST1H4E; MIM: 602830), H4C6 cations of Lys91.18 (HIST1H4F; MIM: 602824), H4C9 (HIST1H4I; MIM: The genetic findings of the cohort are striking, especially 602833), and H4C11 (HIST1H4J) (Figure 1A, Table 1). The given that histones are some of the slowest evolving eu- cohort was collected with trio-based whole-exome karyotic proteins and human H4 is 92% conserved with sequencing in combination with data sharing via Gene- the yeast ortholog.1,2 In the human population, the his- matcher13 and DECIPHER.14 This study received ethical tone H4 genes are tolerant to both loss-of-function and approval from the New Zealand Health and Disability missense variation (gnomAD). We identify nine sites across Ethics Committee (16/STH/3) and London–Riverside REC the 103 amino acid protein with a variant, six of which (09/H0706/20). All families provided consent to be were found recurrently (Pro32, Arg40, Arg45, His75, involved in this project and separate consent was obtained Lys91, and Tyr98), including two where the same site for the use of photos. (Pro32 and Arg40) is altered in multiple different H4 genes. There are fourteen canonical histone H4 genes in the All sites are conserved through to Saccharomyces cerevisiae. human genome, clustering in three genomic loci. At a The altered residues cluster in two main regions of histone nucleotide level, all genes are different, but together H4 (Figure 1B); one cluster centers on the first a-helix of H4 they encode an identical protein. Transcription of these (Figure 1B; purple spheres), a region important for DNA genes is independently regulated, and differing expres- contacts and protein interactions with H3 and histone sion levels are observed during brain development15,16 chaperones.18 Arg45 is positioned in the loop following and in human tissues.4 The genes harboring variants iden- the first a-helix and forms one of the sprockets of the tified in our cohort are among the more highly expressed, nucleosome that contacts the minor groove of DNA. Sub- however, as H4 transcripts are not polyadenylated and stitutions at Arg45 in S. cerevisiae have proven to be delete- therefore missed in most RNA sequencing (RNA-seq) pro- rious to growth and fitness with altered chromatin remod- tocols, limited expression data are available. eling.7,19,20 The second cluster is within the core of the All variants observed were absent from control databases nucleosome (Figure 1B, orange spheres), where important (1KG, gnomAD v2.1.1).17 Furthermore, there were no structural contacts exist between the H3-H4 dimer and missense variants affecting Lys91 in any of the 14 canoni- with histone chaperones.18 Suggestive evidence for these cal histone H4 genes in gnomAD, and only extremely other variants also originates from S. cerevisiae, where a rarely were substitutions observed for the other positions Gly94 mutant (a key residue for H4 C-terminal flexibility) City, MO 64108, USA; 17Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94143, USA; 18Division of Medical Ge- netics, Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94143, USA; 19Department of Clinical Genetics, Leiden Univer- sity Medical Center, 2333 Leiden, the Netherlands; 20Department of Women’s and Children’s Health, Dunedin School of Medicine, University of Otago, Dunedin 9016, New Zealand; 21Guy’s and St Thomas’ NHS Foundation Trust, London SE1 9RT, UK; 22Department of Human Genetics, Radboud University Medical Centre, 6500 HB Nijmegen, the Netherlands; 23Department of Genetics, University Medical Centre Utrecht, 3584 CX Utrecht, the Netherlands; 24 Genetics Laboratory, UDIAT-Centre Diagnòstic, Parc Taulı́ Hospital Universitari, Institut d’Investigació i Innovació Parc Taulı́ I3PT, Universitat Autònoma de Barcelona, 08208 Sabadell, Spain; 25Paediatric Unit, Parc Taulı́ Hospital Universitari, Institut d’Investigació i Innovació Parc Taulı́ I3PT, Universitat Autònoma de Barcelona, 08208 Sabadell, Barcelona, Spain; 26Kaplan Medical Center, Clalit Health Services, Rehovot 76100, Israel; 27Praxis für Humange- netik Tübingen, 72076 Tuebingen, Germany; 28Service of Genetic Medicine, Geneva University Hospitals, 1205 Geneva, Switzerland; 29Department of Ge- netic Medicine, University Hospitals of Geneva and University of Geneva Medical Faculty, Geneva 1211, Switzerland; 30UMR1231 GAD, Inserm – Univer- sité Bourgogne-Franche Comté, 21078 Dijon, France; 31Centre de Référence Déficiences Intellectuelles de Causes Rares, Dijon Bourgogne University Hospital, 21079 Dijon, France; 32Centre de Référence Maladies Rares ‘‘Anomalies du développement et syndromes malformatifs,’’ Centre de Génétique, FHU-TRANSLAD, Dijon Bourgogne University Hospital, 21079 Dijon, France; 33Department of Pediatrics, Naval Medical Center San Diego, San Diego, CA 92134, USA; 34Ambry Genetics, CA 92656, USA; 35Univ. Lille, RADEME EA7364, CHU Lille, Institut de Génétique Médicale, 59000 Lille, France; 36 Department of Clinical Genetics, CHU Lille, 59000 Lille, France; 37Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK; 38Ser- vice de Génétique Médicale, CHU d’Angers, 49933 Angers, France; 39Center for Human Genetics, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, 1200 Brussels, Belgium; 40MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK; 41Rosamund Stone Zander Translational Neuroscience Center, Boston Children’s Hospital, Boston, MA 02115, USA; 42Université de Paris, Department of Clinical Genetics and Reference Centre for Constitutional Bone Diseases, INSERM U1163, Imagine Institute, Necker-Enfants Malades Hospital, AP-HP, 75015 Paris, France; 43Genetic Department, APHP, Sorbonne Université, Pitié-Salpêtrière Hospital, 47-83 Boulevard de l’Hôpital, 75013 Paris, France; 44 CRMR Malformations et Maladies Congénitales du Cervelet et Déficiences Intellectuelles de Causes Rares, Département de Génétique, Sorbonne Univer- sité, AP-HP, Hôpital Trousseau, 75012 Paris, France; 45Area di Ricerca Genetica e Malattie Rare, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146 Rome, Italy; 46Area di Ricerca Medicina Multimodale di Laboratorio, Ospedale Pediatrico Bambino Gesù, IRCCS, 00146 Rome, Italy; 47Area di Ricerca Scienze Neu- rologiche e Medicina Riabilitativa, Ospedale Pediatrico Bambino Gesù, IRCCS, 00163 Rome, Italy; 48Department of Genetics, Necker Enfants Malades Hos- pital, Paris Descartes-Sorbonne Paris Cité University, 75015 Paris, France; 49CHU Lille, Clinique de Génétique, 59000 Lille, France; 50Department of Pedi- atric Cardiology, Division of Pediatrics, University Medical Center Utrecht, 3584 Utrecht, the Netherlands *Correspondence: louise.bicknell@otago.ac.nz (L.S.B.), g.vanhaaften@umcutrecht.nl (G.v.H.) https://doi.org/10.1016/j.ajhg.2022.02.003. The American Journal of Human Genetics 109, 750–758, April 7, 2022 751 Pro32Leu Lys91Gln A Pro32Leu Lys91Gln Pro32Ala Lys91Gln Gly94Arg H4C3 (HIST1H4C) H4C6 (HIST1H4F) Arg40His H4C4 (HIST1H4D) His75Arg Arg40Leu His75Arg Arg40Cys Arg40Cys Arg45Cys H4C9 (HIST1H4I) Arg40Cys Arg45Cys Arg40Cys Arg45Cys Leu37Pro Arg45Cys Arg35Trp Arg45Cys Pro32Arg Arg45Cys Tyr98His Lys31Thr Arg45Cys Tyr98His Arg40Cys H4C5 (HIST1H4E) H4C11 (HIST1H4J) Arg40His Arg40Cys Arg40Cys Arg40Cys Arg40Cys Arg40Leu Arg40Cys Leu37Pro Arg45Cys Arg35Trp Arg45Cys Lys91Gln Pro32Ala Arg45Cys Lys91Gln Pro32Leu Arg45Cys Lys91Gln Pro32Leu Arg45Cys Gly94Arg Pro32Arg Arg45Cys His75Arg Tyr98His Lys31Thr Arg45Cys His75Arg Tyr98His N-TERMINAL TAIL CORE GLOBULAR DOMAIN C-TAIL cluster 1 cluster 2 B H75 K91 Y98 G94 P32 L37 K31 R40 R35 R45 Figure 1. H4 variants identified in the cohort (A) Highly recurrent variants were found in six different H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11), which all encode an identical protein. Aggregate prevalence of disease-causing amino acid changes is also shown. The N-terminal methionine is cleaved from histone H4, and therefore all numbering is relative to the mature polypeptide, in keeping with the protein literature. (B) The affected residues of H4 (orange ribbon) either cluster to the N-terminal a-helix facing toward DNA (cluster 1, purple spheres) or are located in regions buried within the nucleosome core (cluster 2, orange spheres). Size of sphere indicates the relative prevalence of substitutions affecting that residue. 752 The American Journal of Human Genetics 109, 750–758, April 7, 2022 Table 1. Variants identified in H4 genes Number of Chromosomal individuals Gene position (hg38) cDNA Protein H4a numbering CADD PHRED REVEL score 1 H4C3 6: 26,104,044 c.97C>G p.Pro33Ala Pro32Ala 23.1 0.526 2 H4C3 6: 26,104,045 c.98C>T p.Pro33Leu Pro32Leu 25.7 0.438 3 H4C3 6: 26,104,221 c.274A>C p.Lys92Gln Lys91Gln 26.6 0.706 1 H4C4 6: 26,188,955 c.122G>A p.Arg41His Arg40His 23.9 0.459 1 H4C5 6: 26,204,739 c.95A>C p.Lys32Thr Lys31Thr 26.6 0.523 1 H4C5 6: 26,204,742 c.98C>G p.Pro33Arg Pro32Arg 25.3 0.541 1 H4C5 6: 26,204,750 c.106C>T p.Arg36Trp Arg35Trp 26.6 0.494 1 H4C5 6: 26,204,757 c.113T>C p.Leu38Pro Leu37Pro 27.5 0.659 4 H4C5 6: 26,204,765 c.121C>T p.Arg41Cys Arg40Cys 26.6 0.446 7 H4C5 6: 26,204,780 c.136C>T p.Arg46Cys Arg45Cys 26.3 0.589 2 H4C5 6: 26,204,939 c.295T>C p.Tyr99His Tyr98His 26.9 0.317 1 H4C6 6: 26,240,708 c.283G>A p.Gly95Arg Gly94Arg 24.5 0.677 1 H4C9 6: 27,139,430 c.122G>T p.Arg41Leu Arg40Leu 26.7 0.628 2 H4C9 6: 27,139,535 c.227A>G p.His76Arg His75Arg 25.3 0.754 1 H4C11 6: 27,824,245 c.121C>T p.Arg41Cys Arg40Cys 25.8 0.541 RefSeq IDs: GenBank: NM_003542.4 (H4C3), GenBank: NM_003539.4 (H4C4), GenBank: NM_003545.3 (H4C5), GenBank: NM_003540.4 (H4C6), GenBank: NM_003495.2 (H4C9), GenBank: NM_021968.4 (H4C11). a Note on nomenclature: to refer to the residues belonging to this study, HGVS Variant nomenclature would include Methionine-1 (Met1) at the translation initi- ating site, e.g., H4C3 Pro33Ala (c.97C>G [p.Pro33Ala]). However, as the research field of epigenetics and oncohistones typically drops this first post-translation- ally removed methionine, we have also done so. Therefore, the above-mentioned example (included in this study) is referred to as H4C3 Pro32Ala. confers reduced viability,21 whereas a His75 mutant dis- with greyed, thinning hair and wrinkly skin, looking at rupts DNA damage repair processes.22 Other studied vari- least two decades beyond his biological age, which did ants in similar regions (either somatic ‘‘oncohistone’’ vari- not occur in his parents. Premature aging has also been ants in H4,7,8 or recently described germline variants in observed in Rahman syndrome (MIM: 617537), caused H323) are predicted to perturb either nucleosome stability by pathogenic variants in H1-4 (HIST1H1E; MIM: or interaction with histone chaperones, suggesting the 142220).24 This phenotype appears to be milder in our H4 variants described in our cohort most likely cause H4 cohort, however the majority of the individuals are still similar effects. These complementary observations, along- quite young. One individual died from leukemia stemming side the significant recurrence of specific variants, provide from myelodysplasia (P28), but no other individuals were strong evidence for pathogenicity. reported to have bone marrow abnormalities. All individuals displayed intellectual disability (ID) and Non-neurological features were variable across the the majority demonstrated global and/or gross motor cohort. Facial features comprised a wide spectrum developmental delay (Table 2, Table S1). Other neurodeve- (Figure 2B). While some individuals were relatively non- lopmental features such as hypotonia (34%), seizures dysmorphic, others had a common presentation affecting (17%), or autism (17%) were present in some individuals the facial midline, with hypertelorism (17%), a high nasal but less common. Brain MRI was generally normal except bridge (or conversely very low nasal bridge) with a broad for two individuals with delayed myelination. Micro- nasal base (38%) and narrow nares, wide mouth with a cephaly was commonly observed (Figure 2A); an occipito- gap between central incisors or other tooth anomalies frontal circumference Z score smaller than 2 SD was (21%) (Figure 2C), and moderately pointy chin. Visual evident in 16% of individuals at birth and became impairment such as strabismus, astigmatism, or myopia progressively more severe with age (76% at the most recent were reasonably common (61% individuals), and 24% in- exam). Short stature (defined as Z score for height smaller dividuals demonstrated hearing impairment. Skeletal than 2 SD) and failure to thrive were common features development was normal for most individuals, however (38%), however without significant change over time recurring features such as vertebral or digit abnormalities (Figure 2A). Clustering of anthropometric data by H4 were present in several individuals (Figure 2D), and partic- gene or protein region revealed no obvious genotype- ularly severe in individual P28. Variability in clinical phenotype patterns (Figure 2A). The age range of the features and growth was noted even among the seven indi- cohort is between 10 months and 52 years. Interestingly, viduals harboring the same de novo variant in H4C5 encod- the oldest individual shows signs of premature aging ing Arg45Cys (Figure 2B, Figure S1). The American Journal of Human Genetics 109, 750–758, April 7, 2022 753 rior-posterior axis establishment, and tissue necrosis dur- Table 2. Clinical features of individuals in the H4 cohort ing early embryonic development, as these parameters Proportion Clinical feature (percentage) captured the major defects and the cellular toxicity (Fig- ures 3A–3C) observed across all variants analyzed. Pheno- Neurodevelopment typic observation revealed that variants affecting Lys91 Intellectual disability 29/29 (100%) and His75 caused the strongest effects. Interestingly, these Developmental delay 29/29 (100%) two variants both cluster at the core of the nucleosome, and His75 plays a role in DNA damage repair,22 a process Hypotonia 10/29 (34%) previously suggested to be involved in the syndromic fea- Seizures 5/29 (17%) tures of Lys91 individuals.4 His75 is at the interface be- Autism 5/29 (17%) tween two histone molecules within the octamer (in this Ataxia 4/29 (14%) case, H4 and H2B) and it is located in the lrs (loss-of-ribo- somal DNA silencing) domain of H4,22 a nucleosomal sur- Growth face structure reported to have gene-specific silencing Microcephaly—prenatal onset 2/19 (11%) function in yeast.19 Additionally, we observed a dosage- Microcephaly—postnatal 20/29 (69%) dependent effect for Arg40His and Arg45Cys (Figures 3D and 3E), which has been noted for other sprocket arginine Short stature 11/29 (38%) substitutions and may relate to possible roles in higher-or- Failure to thrive 11/29 (38%) der organization of chromatin.25 Interestingly, such Skeletal features sprocket function has been reported for both the sin (switch-independent) and lrs H4 domains containing Craniosynostosis 2/29 (7%) Arg45 and His75, respectively.26 These regions have an Digit anomalies 4/29 (14%) almost identical three-dimensional structure26 and play a Vertebral anomalies 4/27 (15%) role in gene regulation (repression), the perturbation of Facial features which most likely results in pathogenicity and was de- tected in our functional assay. The variability in frequency Hypertelorism 5/29 (17%) of phenotype occurrence across variants may reflect the Upslanting palpebral fissures 3/29 (10%) importance of the affected residues in cellular processes Broad nasal tip 11/29 (38%) crucial to active cell proliferation, as the early zebrafish em- bryo is a system in which cells have a relatively short cell- Thin upper lip/vermillion 4/29 (14%) cycle time. The milder effect observed in our functional Teeth anomalies 6/29 (21%) assay for variants affecting Pro32 and Arg40 may point to Other features a moderate requirement, however the strong recurrence of variants affecting these residues in our cohort provides Recurrent infections 4/29 (14%) alternative evidence to support their pathogenicity. Alto- Visual impairment 17/28 (61%) gether, these results provide a first picture of the variety Hearing impairment 7/29 (24%) of phenotypes caused by the assayed variants, supporting Age range 10 months – 52 years our genetic data. However, as their interpretation is limited (median 10 years 11 months) by their transience and the inherent variability of our cur- rent mRNA assay, further testing in a stable model is required to obtain more insight in the molecular mecha- To validate the pathogenic effect of these variants, we ex- nisms linking genetic variants and phenotype. pressed wild-type human histone H4 and the histone H4 Through genetic and developmental findings, we have variants pertaining to this study in zebrafish by means of identified pathogenic substitutions in six genes encoding mRNA microinjection (Figure 3). The complete conserva- histone H4 in a large cohort of individuals with a neurode- tion of the zebrafish and human histone H4 proteins at velopmental syndrome. Despite well-established cellular the amino acid sequence level and the early, mRNA-medi- requirements for post-translational modification of the ated ectopic overexpression make this an optimal set-up N-terminal tail of H4,27–29 it is notable that no de novo var- for assaying the variants’ effect on early development. iants were identified in this region. Early embryonic effects were evaluated at 28 hours post Recently, a large cohort of individuals with a neurodegen- fertilization (hpf) as fundamental developmental processes erative and developmental disorder were reported harboring such as gastrulation and primary organogenesis are de novo missense variants in the histone H3 replication-inde- completed by this time point. All variants tested displayed pendent genes H3F3A (MIM: 601128) and H3F3B (MIM: significant visible developmental effects compared to 601058).23 While there are phenotypic commonalities be- microinjection of the corresponding wild-type H4 gene, tween the H3.3 cohort and individuals presented here, the except the Pro32Ala and Arg40Cys variants. Specifically, presentations are distinct. The H3F3A/H3F3B cohort appears classification was based on cephalic development, ante- to have a more expansive neurological dysfunction with 754 The American Journal of Human Genetics 109, 750–758, April 7, 2022 A B C P1 Pro32 P3 Pro32 P13 Arg40 P26 Arg40 P15 Arg40 P13 Arg40 P20 Arg45 P6 Lys91 P5 Lys91 P15 Arg40 P21 Arg45 P19 Arg45 P16 Arg45 P21 Arg45 P20 Arg45 P22 Arg45 P25 Gly94 P27 His75 P4 Lys91 P28 His75 P23 Tyr98 P17 Arg45 P16 Arg45 P21 Arg45 P20 Arg45 P22 Arg45 D P1 Pro32 P19 Arg45 P28 His75 P27 His75 P4 Lys91 P6 Lys91 P25 Gly94 P23 Tyr98 P28 His75 P26 Arg40 P21 Arg45 P28 His75 P4 Lys91 P6 Lys91 P25 Gly94 P24 Tyr98 P25 Gly94 P5 Lys91 P6 Lys91 Figure 2. Clinical characteristics of individuals with histone H4 gene variants (A) Individuals with variants in histone H4 genes demonstrate a reduction in height, weight, and brain growth (OFC, occipitofrontal circumference); the latter significantly progresses as the individuals age. There are no detectable genotype-phenotype patterns separating by the specific histone H4 gene or variant cluster. ****p < 0.0001. (B) Facial dysmorphism affecting midline structures is noticeable among the cohort, but highly variable, with no obvious genotype- phenotype correlation. (C) Individuals can present with abnormalities in the appearance and position of teeth (for example, P5, P25). A recurring feature present in several individuals is a noticeable gap between the upper central incisors. (D) Individuals with variants in histone H4 genes also show a spectrum of toe anomalies, ranging from no anomalies present (for example, P21) through to severe 2–3 toe (P1, P28) or 3–4 toe (P25) syndactyly, which can be bilateral. Toes can also be short (P19, P28). anomalies noted on imaging, accompanied by septal or gen- observed is significant, along with the clear clustering of ital abnormalities and craniosynostosis. In contrast, our H4 the variants in two regions of the H4 protein. cohort has ID/DD and microcephaly as presenting features, The redundancy of the H4 genes in the human genome but only more rarely are there other neurodevelopmental is remarkable. Loss-of-function variants in H4 genes are abnormalities. The pathogenic variants identified in H3F3A present in the healthy population17 and are even present and H3F3B are located throughout the protein, with a in homozygous form,30 supporting our hypothesis that smaller number of recurrently occurring variants. In compar- the variants identified here act through a dominant effect. ison, for histone H4, the high level of recurrent variants This disease mechanism, combined with the paralogous The American Journal of Human Genetics 109, 750–758, April 7, 2022 755 Figure 3. H4 variants induce developmental defects in zebrafish embryos (A) Phenotypical characterization in 28 hpf embryos. Representative images of observed phenotypes in zebrafish embryos 28 hpf micro- injected with mRNA encoding either wild-type or identified variants at the one-cell stage. The different classes are defined on general development and necrosis. Arrowhead, cephalic necrosis; arrow, curved tail. (B and C) High magnification examples of cephalic necrosis (B) and curved tail (C) phenotypes. (D and E) Quantification of the phenotypical classification as described in (A). Variants reported in (D) were microinjected with 50 pg/ embryo, and additional testing with 100 pg/embryo is reported in (E). Data marked with a hash symbol was previously published in Tessadori et al.4 Fisher’s exact test: ns, not significant; p > 0.05; *p < 0.05; ****p < 0.0001. Scale bars: 100 mm (A); 50 mm (B and C). landscape of H4 genes, presents opportunities for future by Fondazione Bambino Gesù (Vite Coraggiose), Italian Minis- treatment strategies through targeted knockdown of spe- try of Health (5x1000, CCR-2017-23669081 and RCR-2020- cific H4 gene products. 23670068_001), Italian Ministry of Research (FOE 2019), and the Cliff Broad Family Trust, administered by the Neurological Foundation of New Zealand. The opinions expressed here are Data and code availability those of the authors and do not reflect those of the Navy, the Department of Defense, or the United States government. The Full genetic data are not available due to privacy regulations. DDD study presents independent research commissioned by the Health Innovation Challenge Fund (grant HICF-1009-003), a parallel funding partnership between the Wellcome Trust Supplemental information with the Department of Health and the Wellcome Trust Sanger Supplemental information can be found online at https://doi.org/ Institute (grant WT098051). The views expressed in this publi- 10.1016/j.ajhg.2022.02.003. cation are those of the author(s) and not necessarily those of the Wellcome Trust or the Department of Health. The study has UK Research Ethics Committee approval (10/H0305/83, Acknowledgments granted by the Cambridge South REC, and GEN/284/12, granted The authors thank all individuals and their families who were by the Republic of Ireland REC). The research team acknowl- involved in this study; Marine Tessarech for molecular diag- edges the support of the National Institute for Health Research nostic assistance; Anisha Chopra for experimental work; and through the Comprehensive Clinical Research Network. Jacques Giltay, Meriel McEntagart, and Cynthia Morton for advice. F.T. is supported by NWO grant NWO/OCENW. Declaration of interests GROOT.2019.029, A.O.M.W. was supported by the NIHR Oxford Biomedical Research Centre, K.K. and L.S.B. were sup- The authors declare no competing interests. ported by the Marsden Fund, and L.S.B. was supported by a Rutherford Discovery Fellowship, both administered by Received: November 24, 2021 the Royal Society of New Zealand. A.O.D.L. is supported by a Accepted: February 3, 2022 Manton Endowed Scholar award. Project support was provided Published: February 23, 2022 756 The American Journal of Human Genetics 109, 750–758, April 7, 2022 Web resources 12. Tropberger, P., and Schneider, R. 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